Adjuvant use of laser in eyes with macular retinoblastoma treated with primary intravenous chemotherapy.

BACKGROUND: Adjuvant use of laser with systemic chemotherapy for treatment of retinoblastoma may reduce recurrence rates while also causing local side effects. Information is lacking on the effect of laser on visual outcomes. METHODS: A retrospective review of two retinoblastoma centres in the United Kingdom was conducted. Patients were included if there was a macular tumour in at least one eye. Eyes that received chemotherapy alone were compared with eyes that received chemotherapy plus adjuvant laser. RESULTS: A total of 76 patients and 91 eyes were included in the study. Systemic chemotherapy alone was used in 71 eyes while chemotherapy plus laser was used in 20 eyes. Demographic characteristics of both groups were similar. Macular relapse rates were similar between groups: 22/71 (31%) eyes in chemotherapy group and 9/20 (45%) eyes in laser group (p=0.29). There was no increase in vitreous relapses in the laser group (2/20 eyes), compared with the chemotherapy group 10/71 eyes (p=0.99). Survival analysis demonstrated similar time to first relapse between groups. Final visual acuity was equal between groups with 6/15 or better present in 31.1% of eyes in the chemotherapy group and 37.5% of eyes in the laser group (p=0.76). Presence of tumour at the fovea was predictive of final visual acuity, regardless of treatment group. CONCLUSION: Adjuvant laser in the treatment of retinoblastoma is safe and does not lead to increased rate of vitreous recurrence. Final visual acuity is determined by the presence of tumour at the fovea and not the use of laser.

Isolated Intraocular Relapse of Pediatric B-cell Precursor Acute Lymphoblastic Leukaemia Following Chimeric Antigen Receptor T-lymphocyte Therapy.

Chimeric antigen receptor T-lymphocytes (CAR T) targeting the CD19 surface antigen have achieved a breakthrough in the treatment of multiply relapsed and refractory bone marrow (BM) disease in childhood B-cell precursor acute lymphoblastic leukaemia (B-ALL). The ability of CAR T therapy to treat extramedullary (EM) disease is less proven. However, early reports suggest trafficking of CART-cells to the central nervous system (CNS) as well as other EM sites. We describe a case of isolated intraocular relapse of pediatric B-ALL following CAR T-cell therapy, which had successfully controlled multiply relapsed BM and CNS disease. CAR T-cells may not be able to traffic into the eye, making it a "sanctuary" site during therapy.

The majority of autosomal recessive nanophthalmos and posterior microphthalmia can be attributed to biallelic sequence and structural variants in MFRP and PRSS56.

This study aimed to genetically and clinically characterize a unique cohort of 25 individuals from 21 unrelated families with autosomal recessive nanophthalmos (NNO) and posterior microphthalmia (MCOP) from different ethnicities. An ophthalmological assessment in all families was followed by targeted MFRP and PRSS56 testing in 20 families and whole-genome sequencing in one family. Three families underwent homozygosity mapping using SNP arrays. Eight distinct MFRP mutations were found in 10/21 families (47.6%), five of which are novel including a deletion spanning the 5' untranslated region and the first coding part of exon 1. Most cases harbored homozygous mutations (8/10), while a compound heterozygous and a monoallelic genotype were identified in the remaining ones (2/10). Six distinct PRSS56 mutations were found in 9/21 (42.9%) families, three of which are novel. Similarly, homozygous mutations were found in all but one, leaving 2/21 families (9.5%) without a molecular diagnosis. Clinically, all patients had reduced visual acuity, hyperopia, short axial length and crowded optic discs. Retinitis pigmentosa was observed in 5/10 (50%) of the MFRP group, papillomacular folds in 12/19 (63.2%) of MCOP and in 3/6 (50%) of NNO cases. A considerable phenotypic variability was observed, with no clear genotype-phenotype correlations. Overall, our study represents the largest NNO and MCOP cohort reported to date and provides a genetic diagnosis in 19/21 families (90.5%), including the first MFRP genomic rearrangement, offering opportunities for gene-based therapies in MFRP-associated disease. Finally, our study underscores the importance of sequence and copy number analysis of the MFRP and PRSS56 genes in MCOP and NNO.

Non-invasive diagnosis of retinoblastoma using cell-free DNA from aqueous humour.

Retinoblastoma is the most common eye malignancy in childhood caused by mutations in the RB1 gene. Both alleles of the RB1 gene must be mutated for tumour development. The initial RB1 mutation may be constitutional germline or somatic (originating in one retinal cell only). Distinguishing between these alternative mechanisms is crucial, with wider implications for management of the patient and family members. Bilateral retinoblastoma is nearly always due to a constitutional mutation; however, approximately 15% of unilateral cases also carry a germline mutation, and identifying these cases is important. This can be achieved by identifying both mutation types in tumour tissue and excluding their presence in blood. Modern eye-saving chemotherapy treatment (systemic, intra-arterial and intravitreal) has resulted in fewer enucleations. As a result, tumour tissue required to identify sporadic RB1 mutation(s) is not always available. Modern intravitreal chemotherapeutic techniques for retinoblastoma involve aspiration of aqueous humour (AH), providing a novel sample source for analysis. By analysing cell-free DNA present in the AH fluid of eyes affected with retinoblastoma, we have developed a screening test capable of detecting somatic RB1 mutations that is comparable to current tests on enucleated tumour tissue. The results obtained with fluid from enucleated eyes were concordant with tumour tissue in all 10 cases analysed. In addition, AH analysis from two patients undergoing intravitreal chemotherapy successfully identified somatic variants in both cases. Our findings suggest that AH fluid is a promising source of tumour-derived DNA in retinoblastoma for analysis.

Long-term retinoblastoma follow-up with or without general anaesthesia.

BACKGROUND: Children with treated retinoblastoma undergo frequent examinations to monitor for recurrent or new tumours. Examinations under anaesthesia allow a more complete examination in younger children, however they are stressful for the family, subject the child to medical risk and consume resources. The risk of recurrent or new tumours declines with age and it is common practice to examine older children without general anaesthesia. There are no studies on the safety and cost effectiveness of this practice, or guidelines on when examination without anaesthesia (EWA) can be safely commenced. PROCEDURE: Retrospective case note review of 128 sequential patients treated for retinoblastoma in a national referral centre over 10 years. RESULTS: Following exclusions, 113 eyes of 84 children were analysed. The mean age at diagnosis was 20 months (range birth to 71 months). There were 55 unilateral and 29 bilateral cases. The mean follow-up was 77.7 months (range 12-178 months). EWA was commenced at a mean age of 53 months (range 12-98 months). The age of conversion to EWA was largely dependent on child cooperation and disease activity. Tumour activity was detected on EWA in one child at the age of 86 months, 9 months after the last active treatment and treated successfully. CONCLUSIONS: Examination without general anaesthesia does not appear to expose children to an increased risk of undetected tumour growth. This study highlights the important factors to be considered when deciding a safe time to commence EWA.

Loss-of-function mutations in RAB18 cause Warburg micro syndrome.

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.

A phenotypic variant of Knobloch syndrome.

Knobloch syndrome (KNO) is a rare autosomal recessive condition caused by pathogenic mutations in the COL18A1 gene. It is characterized by high myopia, vitreoretinal degeneration, retinal detachment and midline encephalocoele or midline occipital bone defect. We report a case of KNO confirmed by direct sequence analysis of the COL18A1 gene with typical ocular features, and previously unreported systemic features: occipital hair tuft with transient CSF leak and bilateral renal abnormalities. This case illustrates a new phenotypic variant of this syndrome.

Study of p.N247S KERA mutation in a British family with cornea plana.

PURPOSE: To report clinical and genetic findings in a white British family with autosomal recessive cornea plana (CNA2) with a negative history for consanguinity. To look for evidence of a common ancestry with previously reported Finnish CNA2 patients by studying haplotypes. METHODS: Clinical examination and direct sequencing of the keratocan (KERA) gene was performed in two siblings affected with CNA2 and one unaffected parent. We also studied 22 single nucleotide polymorphisms distributed in the KERA genomic region by direct sequencing in this family as well as in one additional Finnish patient with CNA2 and 24 white British control subjects. RESULTS: Both siblings had the homozygous c.740A>G mutation leading to a p.N247S amino acid change originally reported as the founder mutation in 35 Finnish families. Genetic characterization of genomic regions surrounding the gene revealed large linkage disequilibrium, but the presence of shared extended haplotypes between affected individuals from Finland and the United Kingdom is consistent with a recent common ancestor. CONCLUSIONS: This is the first description of recessive cornea plana in a white British family and it is the second report on the p.N247S change in the KERA gene. Extended haplotype analysis suggests that the two geographically remote occurrences of the c.740A>G mutation may have a common origin.

Cataract associated with type-1 diabetes mellitus in the pediatric population.

PURPOSE: To report 14 cases (28 eyes) of cataract associated with type-1 diabetes mellitus in young children and adolescents. METHODS: Retrospective review of the medical records of 14 patients from seven institutions. All patients under the age of 18 years who met the inclusion criteria of type-1 diabetes mellitus and cataract were included. RESULTS: Mean age at the time of diabetes diagnosis was 9.8 years (range, 0.5-14 years), and mean age at cataract diagnosis was 11.7 years (range, 5-16 years). Two patients presented with cataracts one month before the diagnosis of diabetes; seven after the diagnosis of diabetes; and in five patients the cataract was found at the time the diabetes was diagnosed by the pediatrician. Nineteen out of 23 operated eyes had a best corrected post operative visual acuity of 20/40 or better. Two patients (4 eyes) developed diabetic retinopathy postoperatively. CONCLUSIONS: Although cataract formation in type-1 diabetes is rare, it is treatable and potentially sight-saving in young children and adolescents.

A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8).

Hermansky-Pudlak syndrome (HPS) is genetically heterogeneous, and mutations in seven genes have been reported to cause HPS. Autozygosity mapping studies were undertaken in a large consanguineous family with HPS. Affected individuals displayed features of incomplete oculocutaneous albinism and platelet dysfunction. Skin biopsy demonstrated abnormal aggregates of melanosomes within basal epidermal keratinocytes. A homozygous germline frameshift mutation in BLOC1S3 (p.Gln150ArgfsX75) was identified in all affected individuals. BLOC1S3 mutations have not been previously described in patients with HPS, but BLOC1S3 encodes a subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Mutations in other BLOC-1 subunits have been associated with an HPS phenotype in humans and/or mouse, and a nonsense mutation in the murine orthologue of BLOC1S3 causes the reduced pigmentation (rp) model of HPS. Interestingly, eye pigment formation is reported to be normal in rp, but we found visual defects (nystagmus, iris transilluminancy, foveal hypoplasia, reduced visual acuity, and evidence of optic pathway misrouting) in affected individuals. These findings define a novel form of human HPS (HPS8) and extend genotype-phenotype correlations in HPS.

Identification of novel RPGR ORF15 mutations in X-linked progressive cone-rod dystrophy (XLCORD) families.

PURPOSE: To test the incidence of mutations in RPGR ORF15 in six families with X-linked progressive retinal degeneration (cone-rod dystrophy [XLCORD], macular or cone dystrophy) and to undertake a detailed phenotypic assessment of families in whom ORF15 mutations were identified. METHODS: To amplify and sequence ORF15 in its entirety, a cloning strategy was developed. Families with mutations in ORF15 underwent electrophysiological testing, color vision assessment, color fundus photography, and fundus autofluorescence (AF) imaging. RESULTS: Novel protein truncation mutations were identified in two families. In family A, a 2-bp mutation was identified in ORF15+A1094C G1095T, predicted to result in a truncated protein (E364D/E365X). In family B, a G-to-T transversion (ORF15+1176G>T) resulted in a nonsense mutation (G392X). Characteristics of phenotype in both families included progressive deterioration of central vision and subsequently night vision, mild photophobia, and moderate to high myopia. Ophthalmoscopic abnormalities were generally confined to the macula. A parafoveal ring of increased AF was observed, and electrophysiological evidence of a greater generalized abnormality in cone than rod responses were consistent with a cone-rod dystrophy phenotype. CONCLUSIONS: The cloning strategy for ORF15 facilitated comprehensive sequence analysis in patients. Two families were identified with nonsense mutations, and clinical evaluation revealed them both to have a similar phenotype. The presence of a parafoveal ring of increased AF was an early indicator of affected status in these families. No disease-causing mutations in ORF15 were detected in four other families, suggesting that ORF15 mutations may not be the most common cause of XLCORD.

Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome.

Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of the Rab3 pathway implicated in exocytic release of neurotransmitters and hormones, in 12 families with Micro syndrome. We hypothesize that the underlying pathogenesis of Micro syndrome is a failure of exocytic release of ocular and neurodevelopmental trophic factors.

Progressive cone dystrophy associated with mutation in CNGB3.

PURPOSE: To determine the molecular basis for phenotypic variability in a three-generation consanguineous family containing a single individual with complete achromatopsia and three individuals with progressive cone dystrophy. METHODS: Four affected individuals underwent ophthalmic examination, electrophysiological assessment, color fundus photography, and psychophysical testing. Blood samples were obtained for DNA extraction and mutation screening of the cone-specific cGMP-gated (CNG) channel protein gene CNGB3 was undertaken. RESULTS: The clinical findings in one family member were consistent with a diagnosis of complete achromatopsia, with nystagmus, photophobia, and poor visual acuity from early infancy and complete color-blindness, normal fundi, and absent cone responses with normal rod responses on electroretinography (ERG). Mutation analysis revealed her to be homozygous for the common CNGB3 achromatopsia mutation, 1148delC (Thr383fs). In contrast, the three other symptomatic individuals in the family had findings consistent with progressive cone dystrophy. Their visual problems began later in childhood (ranging from 3 to 14 years of age) and there was evidence of progressive deterioration in cone function. All three had a marked tritanopic color vision defect and fundoscopy revealed bilateral macular atrophy. Electrophysiological testing of these three subjects demonstrated clear evidence of progressive deterioration of cone responses over time; rod responses were normal. All three individuals with this progressive phenotype were found to be compound heterozygotes for the 1148delC (Thr383fs) frameshift mutation and a novel Arg403Gln missense mutation in CNGB3. CONCLUSIONS: Mutations in CNGB3, which have been shown to cause achromatopsia, are now shown to be associated with autosomal recessive progressive cone dystrophy. In this study, a novel Arg403Gln mutation was identified, located in the middle of the pore domain of the cone CNG cation channel beta-subunit, which when associated with the nonsense mutation Thr383fs, resulted in progressive cone dystrophy.